The Picornaviridae family includes a variety of small, non-enveloped, icosahedral viruses with positive-strand RNA genomes1. Many picornaviruses (e.g., rhinoviruses, polioviruses, coxsackieviruses, enterovirus A71, enterovirus D68) that infect humans and cause high morbidity belong to the Enterovirus genus (EV)1. A number of these viruses have been structurally characterized by X-ray crystallography2, 3, 4, 5, establishing the general mechanisms for virus infection and for the development of effective anti-EV therapeutics. Nevertheless, rhinovirus C (RV-C), a newly discovered species among the EVs, remains enigmatic.
RV-C viruses (55 types), together with RV-A and RV-B viruses (˜100 types), are the leading cause of common colds. However, the RV-C lead to more severe respiratory infections among children than any other known rhinoviruses6. In contrast to other RV, the RV-C utilize cadherin related family member 3 (CDHR3) as a cellular receptor7. This childhood asthma susceptibility gene product is expressed in the human lower respiratory tract8. In line with this etiology, RV-Cs cause a significantly higher rate of lower respiratory tract infections in children than in adults9 and are directly associated with childhood asthma exacerbations10. Similar to influenza, RV-C infections peak in winter months. Currently, there are no vaccines or effective antiviral treatments available.
RV-C isolates have been refractory to structural characterization since their discovery in 200611 because of an inability to infect standard tissue culture (e.g., HeLa)12. Only modeled structures, based on amino acid sequence comparisons, have been available to aid biological investigations12, 13, 14. However, with recent advances in direct electron detection15 and image processing approaches16, 17 single-particle cryo-electron microscopy (cryo-EM) has now emerged as a powerful method for determining near atomic resolution (better than 4 Å) structures of macromolecular assemblies18. Cryo-EM requires only limited amount of sample without intensive purification, offering advantages over X-ray crystallography in structural studies of samples that are difficult to produce.
Picornavirus capsids are assembled from 60 copies of biological protomers, each composed of four proteins, VP1, VP2, VP3 and VP42. The three large surface polypeptides, VP1, VP2 and VP3 are folded into eight-stranded antiparallel “jelly rolls.” During the assembly process, autocatalytic cleavage of precursor VP0 into VP2 and VP4 in the presence of viral RNA results in the formation of full infectious virions19. The arrangement of jelly rolls in the virions exhibits pseudo T=3 icosahedral symmetry with an outer diameter of about 300 Å2, 3. The internal surface of the capsid is lined by the 60 copies of VP4. A surface depression or canyon2, encircling each five-fold axis, is frequently the receptor binding site for many EV20. Amino acid residues located on the outer surface of the virus but not specifically within this canyon are typically involved in forming immunogenic sites recognized by neutralizing antibodies. The canyon allows only limited access to these antibodies21. In many EV, a hydrophobic pocket within the VP1 jelly roll and situated underneath the canyon floor is occupied by a fatty-acid like molecule, or “pocket factor,”22, 23 that regulates the conformational states of the virus during cell entry24. Capsid-binding reagents that replace the pocket factor within VP1 are effective antiviral therapeutics against many EV25, but not RV-C14.
In the Examples below we report atomic resolution cryo-EM structures of the full and native empty particles (NEP) of the cell-adapted RV-C15a strain. These structures highlight novel immunogenic surfaces, a probable binding site for the glycosylated CDHR3 receptor molecule and the requirements for antiviral compound resistance. The novel immunogenic peptides identified in the work reported herein are useful targets for therapeutic antibodies and related therapeutics.